CN112062824A - Application of KRP gene in pest control - Google Patents

Abstract

The invention relates to the technical field of genetic engineering, in particular to application of KRP gene in pest control. The invention preliminarily researches the influence of a new transcription factor BmKRP on the reproductive process of female silkworms by constructing the CRISPR/Cas9 knockout strain of the BmKRP of the silkworms and by carrying out CRISPR/Cas9 knockout on the BmKRP of the silkworms^‑/‑Phenotype observation shows that the deletion of BmKRP influences female reproduction of silkworms, so that ovaries become small, oocyte volume becomes small, and the number of eggs laid by adults is reduced; the mutation deletion of the BmKRP is also found to influence the combination of the BmKRP and a reaction element on a BmKr-h1 promoter, so that the BmKr-h1 is not expressed or is expressed in a down-regulation mode, the method has important significance for explaining the molecular mechanism of the silkworm hormone regulation reproduction, has similar regulation and control mechanisms in other insects through research and speculation, provides clues and theoretical guidance for beneficial insect utilization and new target and new strategy of pest control, and has application significance.

Description

Application of KRP gene in pest control

Technical Field

The invention relates to the technical field of genetic engineering, in particular to application of KRP gene in pest control.

Background

Silkworm (Bombyx mori L.) is a lepidopteran myxomatosis insect belonging to the genus invertebrate, the genus Bombyx mori of the family Bombyx of the phylum arthropoda. In the scientific research field, silkworms as important model insects of lepidoptera are widely used for researching the reproductive heredity and metamorphosis development of insects, so that the growth regulation mechanism is mastered and utilized, and the effects of preventing and treating the pests in agriculture and forestry and the like are finally achieved.

The silkworm belongs to a metamorphosis insect, and one generation needs to go through four development stages of completely different forms of eggs, larvae, pupae and imagoes; the stage of transfer of its larvae into pupae requires five molts. The process of silkworm ecdysis and metamorphosis is regulated by complex hormone endocrine in vivo, but ecdysone (Ecdys-lipids) and Juvenile Hormone (JH) play main regulation roles, and the main active ingredient of ecdysone is found to be 20-Hydroxyecdysone (20-Hydroxyecdysone, 20E). The two hormones regulate the metamorphosis development of insects synergistically and play an important role in the growth and reproduction of the insects.

Kr-h1 (Krluppel homolog 1) belongs to the Krluppel family of genes and encodes a gene having 8C atoms₂H₂Zinc finger domain transcription factor. Kr-h1 also plays an important role in the insect reproduction and egg maturation process. Kr-h1 has been found to affect insect reproduction in a number of species through a number of aspects. In female Locusta migratoria (Locusta migratoria), ovarian dysplasia after Kr-h1 RNAi was found, ovaries were significantly smaller than those of the control group, a similar phenomenon was also found in oriental fruit fly (Bactrocera dorsalis), and the number of eggs laid by female oriental fruit fly was found to be significantly reduced. In brown planthopper (Nilaparvata lu)genes) and Aedes aegypti (Aedes aegypti) interfered with the expression of Kr-h1, and a phenomenon of significantly reduced egg laying number was found. In addition, Kr-h1 is more actively expressed during the embryonic development of Drosophila melanogaster (Drosophila melanogaster), Tribolium castaneum (Tribolium castaneum) and the decidua development of postembryonic larvae; after silencing the gene Kr-h1 of brown planthopper nymph, the abnormal ratio of the female external genitalia is found to be obviously increased; kr-h1 from Frankliniella occidentalis has a very high level of transcription at the embryonic stage, indicating that FoKr-h1 plays a regulatory role at the embryonic stage. Kr-h1 also has the function of regulating the growth, development and metamorphosis of insect larvae. Many studies have demonstrated that JH mediates inhibition of biosynthesis and information transduction of 20E through expression of Kr-h1, thereby inhibiting the development process of allergy. In silkworms, BmKr-h1 was also identified as a JH receptor-activated JH primary response gene. It has also been found that Kr-h1, induced by JH, is highly expressed in the prothymus gland producing 20E precursor, and that high levels of Kr-h1 inhibit 20E biosynthesis by reducing the size of the prothymus gland and reducing the expression of steroidogenic genes, thus prolonging larval stages. It can be seen that Kr-h1 plays an important role in inhibiting the metamorphosis development process of insects by JH signal pathway. However, the question of whether 20E can reversely induce the expression of Kr-h1 and the molecular mechanism related to Kr-h1 is not clear at present.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention discovers that 20E can regulate the expression of Kr-h1 gene in the processes of insect growth and development, metamorphosis development, reproduction and the like through a newly identified transcription factor KRP (Kr-h1 regulation protein). The method has important significance for the reproductive research of the silkworms and provides clues for the utilization of beneficial insects by utilizing reproduction and new targets and new strategies for pest control.

The technical scheme of the invention is shown as follows.

The invention provides an application of KRP gene in preparing a medicine for regulating the expression of Kr-h1 gene, wherein the KRP gene comprises a nucleotide sequence capable of being specifically combined with TTATTAA and AATAATCATT in the promoter region of Kr-h1 gene.

According to some embodiments of the invention, the KRP gene is a BmKRP (bomyx mori l.kr-h1 regulation protein) gene.

According to some embodiments of the invention, the nucleotide sequence of the BmKRP gene is as set forth in SEQ ID NO: 1 is shown.

According to some embodiments of the invention, the amino acid sequence of the BmKRP gene is as set forth in SEQ ID NO: 2, respectively.

According to some embodiments of the invention, the modulation is positive modulation.

Studies have shown that JH can inhibit the synthesis of 20E, and 20E can also inhibit the biosynthesis of JH to promote allergic development. Kr-h1 responds not only to JH induction, but also to 20E induction. The inventor researches and discovers that a region-248 nt to-202 nt of a bombyx mori BmKr-h1 promoter is a key region responding to 20E induction, DNA at two ends of the region-248 nt to-202 nt, -248nt to-241 nt (TTATTAA) and-227 nt to-217 nt (AATAATCATT) can be specifically combined with 20E treated BmN nuclear protein, and a transcription factor BmKRP which can be combined with fragments of 248nt to-241 nt and-227 nt to-217 nt on the BmKr-h1 promoter is found on the basis, wherein the N-end of the transcription factor BmKRP contains 3 typical Cys2/His2 zinc finger domains and is positioned in a nucleus; through EMSA experiments, ChIP experiments, intracellular protein overexpression and RNAi experiments, the fact that BmKRP can be combined with a 20E response element (20E cis-regulatory element,20E CRE) on the BmKr-h1 promoter to further activate the transcription expression of BmKr-h1 is further proved. In addition, similar 20E CRE elements (TTATTAA or AATAATCATT) were found in the promoter upstream of the Kr-h1 gene in Aedes aegypti, Apis mellifera, Pieris pisum, Drosophila melanogaster, and Valeriana rubescens. This indicates that the 20E CRE on the Kr-h1 promoter identified in the present invention is highly conserved across multiple insects, suggesting that 20E induces Kr-h1 expression not only is ubiquitous in insects, but the molecular mechanism by which 20E induces Kr-h1 expression may also be the same. In another aspect, the invention also provides the use of KRP gene as an inhibitory or silencing target for preparing an interfering molecule or CRISPR/Cas9 system specifically interfering the expression of harmful insect gene or inhibiting the growth of harmful insects, wherein the KRP gene comprises a nucleotide sequence that can specifically bind to ttattata and AATAATCATT in the promoter region of Kr-h1 gene.

According to some embodiments of the invention, the KRP gene is a BmKRP gene. The nucleotide sequence of the BmKRP is shown as SEQ ID NO: 1 is shown. Further, the amino acid sequence of the BmKRP gene is shown as SEQ ID NO: 2, respectively.

According to some embodiments of the invention, the interfering molecule is a dsRNA, an antisense nucleic acid, a small interfering RNA, a microrna, or a construct capable of expressing or forming said dsRNA, antisense nucleic acid, small interfering RNA, microrna, of the KRP gene or a transcript thereof as an inhibitory or silencing target.

According to some embodiments of the invention, the CRISPR/Cas9 system comprises Cas9 and a sgRNA that specifically targets the KRP gene; wherein, the nucleotide sequence of sgRNA of the specific targeting KRP gene is shown as SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

In another aspect, the present invention provides a use of KRP gene in the preparation of an agent for controlling harmful insects, wherein the KRP gene comprises a nucleotide sequence that can specifically bind to ttattata and AATAATCATT of the promoter region of Kr-h1 gene.

According to some embodiments of the invention, the KRP gene is a BmKRP gene. The nucleotide sequence of the BmKRP is shown as SEQ ID NO: 1 is shown. Further, the amino acid sequence of the BmKRP gene is shown as SEQ ID NO: 2, respectively.

According to some embodiments of the invention, the insect pest is a lepidopteran insect; preferably, the insect pest is a silkworm.

The inventor researches and discovers that the weight of the silkworm pupae is reduced by 18.69% due to the deletion of BmKRP, the length and the width of the silkworm pupae are respectively reduced by 7.31% and 18.61%, the ovary is reduced, the width and the length of an ovum in the ovary are respectively reduced by 11.03% and 9.47%, and the egg laying number is reduced by 44.68%, so that the BmKRP participates in the regulation and control of the development and the generation of the ovary of the silkworm, has important theoretical significance on the reproductive research of the silkworm, and provides clues for the utilization of beneficial insects by utilizing reproduction and the new target and the new strategy of pest control.

The present invention also provides a method for controlling harmful insects, which comprises: down-regulating the expression or activity of KRP gene in insect pests; wherein the KRP gene comprises a nucleotide sequence that can specifically bind to TTATTAA and AATAATCATT of the promoter region of the Kr-h1 gene; preferably, the KRP gene is a BmKRP gene. The nucleotide sequence of the BmKRP gene is shown as SEQ ID NO: 1 is shown. Further, the amino acid sequence of the BmKRP gene is shown as SEQ ID NO: 2, respectively.

According to some embodiments of the invention, the pest insect has a KRP gene in its genome;

according to some embodiments of the invention, the method of down-regulating the expression or activity of the KRP gene in a pest insect comprises: knocking out or silencing the KRP gene in the genome of the pest insect; or transferring a down regulator which down-regulates KRP gene transcription, polypeptide expression or polypeptide activity into the body of the insect.

According to some embodiments of the invention, the insect pest is a lepidopteran insect. Further, the harmful insect is silkworm.

The invention also provides a CRISPR/Cas9 system for knocking out the BmKRP gene, which comprises Cas9 and sgRNA of the BmKRP gene specifically targeted; wherein, the nucleotide sequence of sgRNA of the specific targeting BmKRP gene is shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

The invention also provides application of the CRISPR/Cas9 system for knocking out the BmKRP gene in preparation of a preparation for controlling harmful insects.

The invention has the beneficial effects that:

the CRISPR/Cas9 knockout strain of the silkworm BmKRP is successfully constructed, the influence of a new transcription factor BmKRP on the reproductive process of female silkworms is preliminarily researched, and the influence on the silkworm BmKRP is obtained^-/-Phenotype observation shows that the deletion of BmKRP influences female reproduction of silkworms, so that ovaries become small, oocyte volume becomes small, and the number of eggs laid by adults is reduced; the mutation deletion of the BmKRP is presumed to influence the combination of the BmKRP and a response element on a BmKr-h1 promoter, so that the BmKRP-h 1 is not expressed or is down-regulated, and simultaneously, genes or nutritional phases related to downstream signal pathways can be interferedThe expression of the related pathway gene further influences the development of the silkworm ovary and the generation of the ovum. Not only enriches the understanding of BmKr-h1 on the regulation and control of silkworm reproduction and egg maturation, but also has important significance on the explanation of the molecular mechanism of silkworm hormone regulation and control reproduction (20E → BmKRP → BmKr-h1), provides clues and theoretical guidance for beneficial insect utilization and new targets and new strategies for pest control, and has application significance.

Drawings

FIG. 1 is a structural diagram of an ORF sequence of BmKRP gene;

FIG. 2 is a schematic structural diagram of BmKRP protein;

fig. 3 is an electrophoretogram of sgRNA synthesis;

FIG. 4 shows BmKRP^-/-G₀Sequencing peak diagrams of the PCR products of dead embryo target sequences of generation silkworms;

FIG. 5 shows BmKRP^-/-G₀Detecting the generation mutation;

FIG. 6 shows BmKRP^-/-G₁Detecting the generation mutation;

FIG. 7 shows BmKRP^-/-G₂Detecting the result of the generation heterozygous mutant;

FIG. 8 shows BmKRP^-/-G₂Detecting results of generation homozygous mutants;

FIG. 9 shows wild type and BmKRP^-/-Appearance diagrams of the silkworms at different growth stages;

FIG. 10 shows wild type and BmKRP^-/-The ovarian structure and the ovum incubation condition of the silkworms;

FIG. 11 shows wild type and BmKRP^-/-Performing data statistics on fertilized eggs and P6 pupas of the silkworms;

FIG. 12 shows the regulation of BmKRP protein on the activity of BmKr-h1 promoter;

FIG. 13 is an alignment of Kr-h1 response to 20E induction elements among different insect species.

Detailed Description

The technical solutions and effects of the present invention will be further described and illustrated with reference to the following specific examples, but the present invention is not limited to these specific embodiments. The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available reagents and materials unless otherwise specified.

Insects: silkworm P50 strain is selected as experimental material. After the silkworm moths lay eggs, firstly, carrying out virus detection treatment, and placing the silkworm moths qualified for virus detection at the temperature of 26 ℃, the relative humidity of 70 +/-5 percent and the illumination of 12: 12(Light: Dark) and hatched larvae were fed with fresh mulberry leaves. Under the condition, the laid silkworm eggs begin to turn green 7 days after egg laying, turn green 8 days later and hatch larvae 9 days later, which are 1-year-old larvae and are also called as newly-hatched silkworms. The newly-hatched silkworms enter 2 ages after 3 days, the interval between 2 ages and 3 ages, and between 3 ages and 4 ages is 3 days, the silkworms begin to molt and enter 5 ages after 4 days of 4 ages, the silkworms begin to stop eating after eating about 6 to 7 days of mulberry leaves and enter a mature silkworm period, the silkworms gradually discharge the residual food in the intestinal tract, the silkworms become transparent, start to spin about half a day, spin on a spinning net and form cocoons for about 2 days, then enter a pre-pupation period, molt and enter the pupation period after lasting about half a day, and the silkworms emerge into adults after 8 to 9 days of pupation period. In order to keep the development period of the taken material to be higher in consistency, a small side of the cocoon is cut off by scissors in the prepupa stage, so that the prepupa state and the pupation time can be conveniently observed and determined.

The primers referred to in the examples were all synthesized by the Shanghai Invitrogen company; the enzymes involved were purchased from TAKARA; cas9 protein was purchased from PNA BIO INC (California, USA); the rest reagents are analytically pure reagents.

The sequences of the primers used in the examples are shown in Table 1.

TABLE 1 primer sequences

Example 1 bioinformatic analysis of BmKRP

The access number of BmKRP in NCBI's Genbank database is XM-004922171.3 (https:// www.ncbi.nlm.nih.gov/nuccore/XM-004922171.3 /), and there is no description or article about the gene in NCBI's database, and the encoded protein is currently an unknown protein. The nucleotide sequence of BmKRP is analyzed to find that the total length of BmKRP gene is 2689bp, the open reading frame is 1557bp, 3 exons and 2 introns are contained, the sizes of the 3 exons are 422bp, 237bp and 899bp respectively, and the sizes of the 2 introns are 1195bp and 1889bp (shown in figure 1). The protein encoded by BmKRP has 3 typical Cys2/His2 zinc finger domains at the N-terminal and 3 low complexity regions at the C-terminal (as shown in FIG. 2). The zinc finger domain is presumed to be a domain binding to DNA as a transcription factor BmKRP. The BmKRP protein encodes 518 amino acids, the predicted molecular weight is 58.951kDa, and the isoelectric point pI is 9.25.

Example 2 design and Synthesis of sgRNAs

(1) Primer design

According to the principle of target design, an online target design program is used for designing a sgRNA core sequence, a site1 is selected as a target site (1015 nt-1037nt of a nucleotide sequence shown by SEQ ID NO: 1 is site1), and sequencing verification shows that all the used P50 bombyx mori BmKRP target site sequences are homozygous and can be used as a target site of Cas9 action.

(2) Primer PCR

The designed forward and reverse primers are sent to a company for synthesis, and are mutually primer amplified through a PCR program to synthesize a complete sgRNA sequence transcription template. The primer sequences used for synthesizing the BmKRP target sgRNA are shown in Table 1 (SEQ ID NO: 3-4). The PCR system used is shown in Table 2, and the amplification procedure is: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, renaturation at 55 ℃ for 15s, extension at 72 ℃ for 20s, 29 cycles; re-extension for 2min at 72 deg.C; storing at 12 deg.C.

Table 2 PCR system for sgRNA synthesis

(3) Construction of sgRNA cloning vector

The PCR product was recovered by cutting the gel and ligated into the pMD-18T cloning vector of TAKARA, as shown in Table 3. The resulting plasmid was transformed into DH 5. alpha. competent cells (purchased from Shenzhen Kangsheng Life technologies, Ltd., catalog No. KTSM 101L).

TABLE 3 pMD18/19-T vector cloning System

Reagent	System/. mu.L
		DNA	4.5
Buffer solution	5
		PMD-18/19T	0.5
Total volume	10

(4) Colony PCR detection of clones reverse inserted into vector

Monoclonal colonies were picked. Colony PCR was performed using pMD-T-F (SEQ ID NO: 7) and R20(SEQ ID NO: 8) as primers, as shown in Table 4, using PCR systems according to the reaction procedure described in (2) primer PCR of example 2, followed by sequencing.

TABLE 4 PCR System for colony PCR

(5) Selecting non-mutated monoclonal flora to extract plasmid

Plasmid extraction was performed using a small and medium Plasmid extraction Kit (TIAN prep Mini Plasmid Kit II).

(6) Preparation of sgRNA in vitro transcription template

Using plasmid without mutation as template, colony PCR primer pMD-T-F (SEQ ID NO: 7) and R20(SEQ ID NO: 8) to amplify a fragment of about 500bp as final in vitro transcription template, the PCR reaction system is shown in Table 5;

TABLE 5 plasmid PCR System

Reagent	System/. mu.L
		Plasmid template	3ng
DNA polymerase	0.3
		10 XPCR buffer	3
pMD-T-F	1
		R20	1
dNTP mix (2.5mM)	0.6
		Addition of ddH2To total volume of O	30

PCR reaction procedure As primer PCR (2) in example 2, PCR product was purified by phenol chloroform (pH >7), and after purification, the precipitate was dissolved in Nuclean-free water, 1. mu.L was used for concentration determination, and 1. mu.L was used for electrophoresis detection.

(7) In vitro synthesis of sgrnas

Using purified PCR product as template and application

And (3) carrying out in-vitro transcription by using the Kit, then purifying by using a phenol chloroform isoamylol method to obtain sgRNA required by injection, taking 1 mu L of sgRNA for electrophoretic detection, and placing the rest in a refrigerator at the temperature of-80 ℃ for later use. The results are shown in FIG. 3, wherein, from left to right, the M lane, lane 1 and lane 2 are shown; m: the DL2000 Marker is 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom in sequence; lane 1 is prepared sgRNA in vitro transcription template, and

the 500bp required for the synthesis of Kit is consistent; lane 2 is synthetic sgRNA.

Example 3 construction of CRISPR/Cas9 knockout line of Bombyx mori BmKRP-/-

1. Experimental methods

1) Screening of silkworm BmKRP mutant

Detection of injection positivity

Injection amount: a proper amount of prepared sgRNA and purchased Cas9 protein are taken to prepare 5 mu L of mixed solution (the final concentration of the sgRNA and the final concentration of the Cas9 are both 600 ng/mu L), and the sgRNA is selected to be mixed and then injected into silkworm eggs together with the Cas9 protein. Cas9 protein 2.5 mu L, site1 sgRNA 2.5 mu L is mixed evenly and injected, and the injection amount is about 2-3nL per egg.

After incubation with injected eggs, the genome is extracted by collecting a mixture of multiple dead embryos and the extracted genomic DNA (extracted conventionally) is treated with ddH₂Diluting the mixture to 100 ng/mu L, taking 1 mu L of dead embryo genome DNA as a template in a 20 mu L PCR system, adding a template/primer mixed solution according to the table 7, mixing the mixed solution uniformly, and centrifuging the mixed solution to the bottom of a tube for carrying out PCR reaction.

And directly sequencing the PCR product after electrophoresis detection, and if a multi-layer peak appears on a sequencing peak image, indicating that the injected silkworm eggs have DNA mutation of the target site.

TABLE 7 PCR System for genomic DNA

Reagent	System/. mu.L
		Genomic DNA
	1
		DNA polymerase	0.2
dNTP	0.5
		10 XPCR buffer	2
Primers SEQ ID NO: 5	0.5
		Primers SEQ ID NO: 6	0.5
ddH2O	Is supplemented to 20

PCR reaction procedure was conducted by referring to (2) primer PCR in example 2. After the reaction, the detection was carried out by 1% agarose electrophoresis.

2)BmKRP^-/- G₀Detection of moth substitutes

Before the specific phenotype of the mutant is not screened, G is ensured₀Complete and normal growth of the surviving individuals, so G can be extracted₀Carrying out PCR on silkworm slough genomes when 5 th generation silkworms pupate, carrying out electrophoresis detection on PCR products, and then sending to sequencing, wherein if a sequencing peak diagram of the PCR products shows double peaks, mutation of DNA of a target site is shown, and if the double peaks do not appear, the DNA still remains wild. And returning the PCR sample with mutation, performing TA-clone, connecting T vectors, separating mixed mutation one by one, then converting, coating a plate, selecting positive monoclone for PCR, carrying out electrophoresis detection, sending to sequencing to obtain each specific mutation type, and comparing with a wild type target sequence to obtain the specific DNA base insertion or deletion condition.

The extraction procedure of genomic DNA and the PCR reaction system are described in section "1) detection of injection positivity".

A connection reaction step: according to the TAKARA product description.

The resulting plasmid was transformed into DH5 α competent cells.

A single colony was picked from an LB plate with ampicillin resistance cultured overnight, and the reaction solution was prepared and mixed thoroughly in the PCR reaction system referred to as "1) test injection positivity".

PCR reaction procedure was conducted by referring to (2) primer PCR in example 2. After the reaction, the detection was carried out by 1% agarose electrophoresis. The PCR product in which the specific DNA band appeared was sent for sequencing.

3) Moth mating strategy

For the injected moth (G)₀Generations), after sequencing, preferentially selecting bimodal heterozygous mutant as parent to carry out mating passage, then extracting silkworm slough genome DNA of pupated 5-year silkworms in each generation, according to G in 2)₀And (3) selecting male and female individuals with double peaks for mating until homozygous mutants appear in the step of moth generation detection.

4)BmKRP^-/-Homozygous mutant screening and phenotypic analysis

Since it has not been determined which aspect is affected by the deletion of Bombyx mori BmKRPFunctionally, it is possible that no visible appearance phenotype appears, but based on the high level expression of BmKRP in the ovary and the RNA interference approach to reduce the pupal BmKr-h1 (gene involved in the regulation of expression of BmKRP) expression, we speculate that BmKRP may be associated with the reproductive development of silkworms. Thus, in addition to the appearance, BmKRP will also be observed^-/-Ovary development and spawning conditions in the homozygous mutant.

2. Results of the experiment

1) Detection of injection positivity

The injected dead embryos were detected by sequencing and the results are shown in FIG. 4 as BmKRP G₀A sequencing peak diagram of a PCR product of a dead embryo target sequence of silkworm generation is underlined, and a BmKRP target sequence is indicated, so that overlapping peaks appear at the target points of the peak diagram, which indicates that mutation occurs, and the synthetic sgRNA can accurately exert a cutting effect.

2)BmKRP^-/- G₀Generation mutant screening

Extracting incubated G₀PCR is carried out on silkworm slough genomes after pupation of all 5-year-old silkworms, and PCR products are directly sent for sequencing. The sequencing result is shown in figure 5, and it can be seen that a double-peak phenomenon appears near the target point, which indicates that the silkworm successfully obtaining the mutant gene can be subjected to homozygous mutant screening. G in which a part of the target sites are mutated₀Mating male and female moths, and part G₀Mating the generation mutant with wild silkworm, collecting silkworm eggs, hatching and preparing for screening homozygous mutant.

3)BmKRP^-/- G₁Generation mutant screening

Normal feeding G₁Standing for larva, and extracting G after it enters pupa stage₁PCR is carried out on silkworm slough genomes when all 5-year-old silkworms pupate, PCR products are directly sent to be sequenced, the sequencing result is shown in figure 6, a bimodal phenomenon appears at the designed position of a target site, and the result shows that the silkworms with mutant genes can successfully enter a pupation stage, and the next mutant screening can be carried out. Selecting and returning sample with double peaks in sequencing, connecting with T carrier, coating plate for later transformation, selecting positive monoclonal for PCR, and detecting by electrophoresisSending out the sequencing, comparing the sequencing result of the mutant genotype with the wild type target sequence on a website on an NCBI BLASTN line to obtain G₁The genotypes of the generation mutant silkworms are shown in table 8. Mutations were found to occur at site 1. The mutant silkworms containing the same genotype were mated and further screened for homozygous mutants.

TABLE 8G₁Genotype of generation mutant silkworm

Note: the target sequence is the bold part, PAM underlines marks, short lines indicate deletion of bases, italic marks indicate insertion of bases, and deletion and the number of inserted bases are marked on the rightmost side (+, insertion; -, deletion).

4)BmKRP^-/- G₂Generation mutant screening

At G₁Selecting parents carrying the same mutant gene, crossing, and mating the parents to generate offspring (G)₂) It is very likely that there will be homozygous mutants. Extraction of G₂PCR is carried out on silkworm slough genomes when all 5-year-old silkworms pupate, the PCR products are sent to be sequenced after electrophoresis detection, the sequencing result is shown in figure 7, double peaks appear at the designed position of a target site, the silkworms are indicated to be heterozygous mutants, the other sequencing result is shown in figure 8, single peaks appear at the designed position of the target site, 4bp is deleted at the target site, the silkworms are indicated to be homozygous mutants, and BmKRP is successfully obtained^-/-The target site lacks 4bp homozygous mutant, and the mutant genotype is as follows: AAAATGGGTTCAA- - -ACCAGG (-4), "-" indicates deletion.

5) Silkworm BmKRP^-/-Phenotypic analysis

Will be at G₂Inbreeding a generation-obtained homozygous mutant lacking 4bp as a parent to obtain G₃Generation silkworms, according to Mendelian's Law of inheritance, G₃The generation individual is also a homozygous mutant with a target point sequence deleted by 4bp, so that the construction of the CRISPR/Cas9 knockout strain of the bombyx mori BmKRP is successful. Will be provided withFertilized egg and G of wild silkworm₃And synchronously and separately incubating fertilized eggs of the generation mutants, and observing the phenotype of the mutant homozygote.

①BmKRP^-/-Phenotypic observations at different growth stages

FIG. 9 shows wild type and BmKRP^-/-Appearance of silkworms at different stages of growth, where Wt denotes wild type, Bmkrp^-/-Represents a BmKRP homozygous mutant; by the pair BmKRP^-/-The appearance and shape of the mutant are observed at each growth stage, and the mutant is not obviously different from the wild type in appearance and shape and slightly different in shape. After egg laying, the homozygous mutant was observed to have eggs that were morphologically indistinguishable from the wild type, but the number of eggs laid by the mutant was relatively small (a-b in FIG. 9); observing 1 st (c in fig. 9) and 5 th (d in fig. 9) larvae of the mutant and wild type, which have no obvious difference in body type and appearance; the homozygous mutant and the wild type silkworm have no obvious difference in appearance at the 6 th day of the pupal stage, but the pupal volume of the homozygous mutant is obviously smaller (e in fig. 9); the homozygous mutant silkworms in the adult stage had slightly smaller body types than the wild type, and were not significantly different in appearance (f in fig. 9). The results show that BmKRP may act on the pupal stage and the adult stage of the silkworm.

②BmKRP^-/-Observation of phenotype associated with ovum development and reproduction

The above results show that BmKRP is compared with wild type^-/-There are differences between pupae and adults. To further understand the effect of BmKRP mutation on silkworms, the imagoes were dissected, and the results are shown in FIG. 10, which is the ovarian structure and ovum incubation condition of wild-type and homozygous mutant silkworms, wherein Wt represents wild-type, Bmkrp^-/-Showing BmKRP homozygous mutant, (A) the ovary structure of silkworm in adult period; (B) arranging eggs in a single egg tube; (C) fertilized eggs on day 8 after spawning; (D) newly hatched larvae. It can be seen that BmKRP is present in comparison with the wild type^-/-The ovaries of (a) were significantly shortened, and the number of ova contained therein was also decreased, but the morphology of the ova was not significantly changed (a in fig. 10); the ovum tubes in the ovary are separated one by one, the ovum shape in the single ovum tube is observed, and BmKRP can be seen compared with wild silkworm^-/-The number of eggs within a single egg tube was significantly reduced (B in fig. 10); observing fertilized egg, BmKRP, at day 8 after spawning^-/-And the eggs laid by the wild-type adults were mostly able to successfully enter the transgenic stage, which indicates that the mutation of BmKRP had no significant effect on the embryonic development of fertilized eggs (C in fig. 10); there was also no significant difference between the mutant and wild type newly hatched larvae (D in fig. 10). The above results preliminarily indicate that BmKRP may be involved in female reproduction, mainly including affecting ovarian development and oogenesis.

③BmKRP^-/-Analysis of pupae, ovum and number of eggs laid

Except for observing BmKRP^-/-Appearance and ovarian development, we also analyzed BmKRP by t-test statistics^-/-The differences from the wild type day 6 pupa body weight, pupa length and width, length and width of eggs in the adult stage and the number of eggs laid by the adult are shown in FIG. 11, in which (A) the pupa weight at day 6 in the pupal stage; (B) the length of pupae on the sixth day of pupal stage; (C) width of pupa on 6 th day of pupal stage; (D) the number of eggs laid by the imagoes; (E) the length of the egg; (F) width of the egg. P6: 6 th day of pupal stage; wt: a wild type; bmkrp^-/-: BmKRP homozygous mutant. "+" denotes p<0.05, ". indicates p<0.01, ". indicates p < 0.001, and the significance statistics were determined by t-test. The results show that BmKRP is compared with wild type^-/-The weight, length and width of pupae were reduced at day 6 of pupal stage (A-C in FIG. 11), by 18.69%, 7.31% and 18.61%, respectively, indicating that the absence of BmKRP affected normal growth at the end of pupal stage. Simultaneous discovery of BmKRP^-/-The egg laying number was reduced 44.68% compared to the wild type (D in fig. 11); statistics of the length and width of the unmatched eggs in the adult stage also show that BmKRP^-/-The reduction was 11.03% and 9.47% compared to wild type (E-F in FIG. 11). The mutation of BmKRP is shown to influence the number of eggs of the silkworm imagoes and the egg maturation process. As the terminal stage of silkworm pupae is the key stage of in vivo ovum maturation, the BmKRP is preliminarily deduced to ensure the normal ovum generation and ovum maturation of the adult silkworm by maintaining the normal growth of the terminal stage of the silkworm pupae.

Example 4 Effect of BmKRP on the BmKr-h1 promoter

BmKRP dsRNA was synthesized using a dsRNA synthesis kit (T7 RiboMAXTM Express RNAi System, available from Promega corporation) and two pairs of primers (SEQ ID NOS: 9-12) having a T7 promoter.

In order to detect the effect of BmKRP protein on BmKr-h1 promoter, a BmKRP-EGFP overexpression vector (taking an EGFP vector as a control) and WT-BmKr-h1-p-Luc plasmid or Mut-BmKr-h1-p-Luc plasmid (mutation site is-248 nt to-217 nt) and an internal reference vector pRL-SV40 transfect BmN cells together, and the activity of luciferase in the cells is detected after 48 h; wherein, BmKr-h1-p-Luc represents a recombinant vector of the BmKr-h1 promoter sequence and pGL3, WT represents that the promoter sequence is wild type, and Mut represents a mutant type of 20E CRE locus on a mutant promoter.

To investigate whether inhibition of BmKRP protein expression in BmN cells would reduce induction of BmKr-h1 promoter by 20E, BmKRP dsRNA was transfected into BmN cells for 24h, with GFP as a control, and primer sequences for synthesizing GFP dsRNA were as shown in SEQ ID NO: 13-16, and carrying out qRT-PCR (primer sequences shown as SEQ ID NO: 17-18) to detect the expression condition of each gene.

After 24h of transfection of BmKRP dsRNA (EGFP dsRNA as control) in cells, WT-BmKr-h1-p-Luc plasmid or Mut-BmKr-h1-p-Luc plasmid and internal reference vector pRL-SV40 were co-transfected into BmN cells, treated with 20E at a final concentration of 1. mu.M (DMSO treatment as control) after 6-8h, and luciferase activity in cells was measured after 48 h.

As shown in fig. 12, is the regulation of the activity of BmKr-h1 promoter by BmKRP protein; a and b indicate that BmKRP and EGFP proteins can be successfully overexpressed in BmN cells and the expression amount is equivalent. B shows that the luciferase activity in the cells co-transfected with the BmKRP-EGFP and WT-BmKr-h1-p-Luc vectors is remarkably enhanced compared with the co-transfected EGFP and WT-BmKr-h1-p-Luc vectors in the cells; the luciferase activity in the cell co-transfected with the EGFP and the mutant BmKr-h1-p-2877bp-Mut-pGL3 vector has no obvious change with the luciferase activity in the cell co-transfected with the BmKRP-EGFP and Mut-BmKr-h1-p-Luc vector. This result indicates that the BmKRP protein activates the BmKr-h1 promoter to transcribe the downstream gene through sites-248 nt to-217 nt on the BmKr-h1 promoter. As can be seen in the C picture, after the dsRNA is transfected, the expression amount of BmKRP is obviously reduced compared with the control, which indicates that the synthesized dsRNA can obviously inhibit the expression of BmKRP. As can be seen in the D graph, 20E had significant activation of the BmKr-h1 promoter when EGFP dsRNA and WT-BmKr-h1-p-Luc were co-transfected, whereas 20E had no significant activation of the BmKr-h1 promoter when BmKRP dsRNA and WT-BmKr-h1-p-Luc were co-transfected; in the experimental group transfected with the Mut-BmKr-h1-p-Luc plasmid, 20E did not significantly activate the mutated promoter, either when transfected with EGFP dsRNA or BmKRP dsRNA.

The above results indicate that reducing BmKRP in BmN cells inhibits the response of BmKR-h1 promoter to 20E, and that 20E activates BmKR-h1 promoter by regulating the binding of BmKRP protein to-248 nt to-217 nt sites of BmKr-h1 promoter.

Example 5 alignment of Kr-h1 response to 20E inducible elements in different insects

From the results of the above examples, it was found that the-248 nt to-217 nt region of the promoter of BmKr-h1 is CRE in response to 20E. It was further found that the-248 nt to-241 nt (TTATTAA) and-227 nt to-217 nt (AATAATATATTATT) regions of the-248 nt to-217 nt region are sites required for binding to nuclear proteins.

20E treatment induced Kr-h1 expression in the larval cuticles of Drosophila melanogaster, Tripsar erythropolis TcA cells, Helicoverpa armigera (Pecase et al., 2000; Xu et al., 2018; Zhang et al.,2018), suggesting that 20E induced Kr-h1 expression may be a common phenomenon in insects. Therefore, we searched TTATTAA (-248nt to-241 nt) and AATAATCATT (-227nt to-217 nt) on BmKr-h1 promoter, which responded to 20E and bound to nucleoprotein, for Kr-h1 promoter sequences of Aedes aegypti, Apis mellifera, Pisum sativum, Drosophila melanogaster and Trimerella erythropolis to analyze whether similar elements were present on the Kr-h1 promoter of these insects.

The results of the analysis showed that similar 20E CRE elements (TTATTAA or AATAATCATT) were found in the Kr-h1 promoters from Aedes aegypti, Apis mellifera, Piper pisum, Drosophila melanogaster and Tripsammophila rubescens species

FIG. 13 shows an alignment of Kr-h1 response to 20E-induced elements in different insect species, in which Aa: aedes aegypti, Aedes aegypti; am, and (2): apis mellifera, Apis mellifera; ap: acrythosphon pisum, pythium pisum; bm: bombyx mori, Bombyx mori; dm: drosophila melanogaster, Drosophila melanogaster; tc: tribolium castaneum, Tribolium castaneum; numbers indicate the distance from the transcription start site (AaKr-h1, BmKr-h1, TcKr-h1, AmKr-h1 and DmKr-h1) or the translation start site (ApKr-h 1); the A diagram is the alignment of the-227 nt to-217 nt region (AATAATCATT), the B diagram is the alignment of the-248 nt to-241 nt (TTATTAA) region sequence, and it can be seen that the-248 nt to-241 nt (TTATTAA) region sequence and the AATAAT in the-227 nt to-217 nt region (AATAATCATT) are completely consistent in each species, and the two regions are adjacent or partially overlapped. This indicates that the 20E CRE on the Kr-h1 promoter we identified is highly conserved across multiple insects, suggesting that not only is the 20E-induced Kr-h1 expression ubiquitous in insects, but the molecular mechanism by which 20E-induced Kr-h1 expression may be the same.

Example 5 BmKRP Gene in different insects

As Kr-h1 of various insects can be induced by 20E, and BmKr-h1 (Gene ID:100415910 at NCBI) with high similarity can be found on Kr-h1 promoters of various insects, and the regions (AATAATCATT) of-248 nt to-241 nt (TTATTAA) and-227 nt to-217 nt of sites responding to 20E induction can be found. The-248 nt to-241 nt (TTATTAA) and-227 nt to-217 nt regions (AATAATCATT) of the BmKR-h1 promoter can be specifically combined with the BmKRP protein.

To determine whether homologous proteins of BmKRP are present in other insects, we first aligned BmKRP At NCBI to homologous genes of BmKRP in other insects, including lepidopteran (Lepidoptera) bollworm (h.armigera, Ha), helicoverpa virescens (Hv), navel orange borer (At), hemimorphia heterophylla (Ba), golden vein butterfly (Danaus plexippus, Dp), golden butterfly (papiio machaon, Pm), phoenix butterfly (papiio, Pp), butterfly (Pieris rapae, Pr), diamond back moth (p.xylella, Px) and prodenia litura (s.litura), and analyzed their amino acid similarities. The results showed that the amino acid similarity of BmKRP to these insect homologous genes was around 30%, that of bombyx mori (gene No. LOC101738779) to navel orange borer (gene No. LOC106134402), hemiptera heteropolaris (gene No. LOC112056002), golden spotted butterfly (gene No. OWR50023.1), cotton bollworm (gene No. LOC110381305), cotton bollworm (gene No. B5V51_576), butterfly of phoenix species (gene No. LOC106108092), butterfly of cabbage (gene No. LOC110997796), diamond back moth (gene No. LOC105380729), butterfly of phoenix species (gene No. LOC106712588) and spodoptera litura (gene No. L0C111360983), that of similarity was 33.8%, 30.3%, 29.2%, 29.5%, 32.0%, 28.6%, 28.8%, 22.2%, 24.7% and that of these insect homologous genes were of small protein species with different structural domains (small protein and large differences from those of these insect species).

TABLE 2 different insects KRP amino acid homology (%)

Note: at: amylois transitella, navel orange borer; ba: biculus anynana, hemimorphous eye butterfly; bm: bombyx mori, Bombyx mori; and Dp: danaus plexippus, golden black-vein butterfly; ha: helicoverpa armigera, cotton bollworm; hv: heliothis virescens, cotton bollworm; pm: papiio machaon, pteris punctata; pp: papiio polypeptides, Papilio ptera leucorrhea; pr: pieris rapae, Pieris brassicae; px: plutella xylostella, Plutella xylostella; pm: papiio machaon, pteris punctata; sl: spodoptera litura, Spodoptera litura.

Although the similarity of BmKRP homologous genes in different insects is not very high, after the amino acid sequence alignment analysis of the BmKRP homologous genes in different insects, the protein is found to be moderately conserved among different insects and all contain typical Cys2/His2 zinc finger domains, but the number of Cys2/His2 zinc finger domains is inconsistent, the protein is moderately conserved in amino acids in 3 zinc finger domains at the N-end, and the terminal 20 amino acids are highly conserved. This result suggests that Cys2/His2 zinc finger domain and the terminal 20 amino acids conserved the function of the protein.

In summary, the present invention is in G₀Generation adopts mutant heterozygote as parent mating to generate G₁Generation, thereby G₁The probability of selecting compound heterozygote (compound heterozygote) is greatly improved. Composite heterozygotes here mean that both alleles have undergone mutation, which is random and therefore of different types, and the bases deleted and inserted are random, and thus are sense heterozygotes, similar to mutant homozygotes. This can be used as a method for rapidly obtaining homozygous mutants.

In pair G₁When the generation silkworm slough genome is sequenced and screened, more sense heterozygous mutations are identified and screened, the compound heterozygous mutants with the same mutation type are mated by testing the genotype, and the offspring G of the hybrid heterozygous mutants₂More homozygous mutants of the same type appear in the mutant, and mainly comprise homozygous mutants which are respectively deleted for 1bp and 4bp at target sites and (-1, -4) compound heterozygous mutations. In the invention, G₂Inbreeding a small number of homozygous mutants with deletion of 4bp in the generation to produce homozygous mutants G with deletion of 4bp₃Therefore, G can be directly observed₃The phenotype of the generation homozygous mutant greatly improves the screening efficiency of the homozygous mutant.

The CRISPR/Cas9 technology is utilized to successfully construct the CRISPR/Cas9 knockout strain of the bombyx mori BmKRP, and a foundation is laid for analyzing the phenotype of a homozygous mutant and exploring the function of the BmKRP. In the experiment, a BmKRP gene knockout target site is also designed, and the result shows that the success rate of the CRISPR/Cas9 system in site1 cleavage is higher.

Through the comparison of silkworm BmKRP^-/-Phenotypic observation shows that the deletion of BmKRP affects female reproduction of silkworm, resulting in diminished ovary, diminished oocyte volume and diminished egg number of adult, which may also result in BmKRP^-/-The reason why the body types at both pupa and adult stages are smaller than the wild type. Therefore, BmKRP is mainly involved in regulating and controlling the development of the ovary and the generation of the ovum of the silkworm.

The BmKr-h1 has higher-level expression in the ovaries at the terminal pupal stage and the adult stage, and the terminal pupal stage is the key stage of egg maturation in the silkworm, suggesting that the BmKr-h1 may participate in the egg maturation process of the silkworm. And the RNAi is utilized to reduce the expression of BmKr-h1 (gene of BmKRP involved in regulating expression) in the pupal stage, so that the maturation of partial oocytes in the ovary is influenced. BmKRP is a novel transcription factor discovered during the identification of proteins that bind to the 20E-responsive element on the BmKr-h1 promoter, and is capable of activating transcriptional expression of BmKr-h 1. In the invention, after BmKRP is mutated by using CRISPR/Cas9, the phenomena of ovary reduction, ovum reduction, remarkable decrease of the egg laying number of adults and the like occur. Therefore, it is speculated that the mutation deletion of the BmKRP affects the combination of the BmKRP and a response element on the BmKr-h1 promoter, so that the BmKr-h1 does not express or down-regulates the expression, and simultaneously possibly interferes the expression of a downstream signal pathway related gene or a nutrition related pathway gene, thereby affecting the development of the silkworm ovary and the generation of the ovum. This is the first time to explore the function of newly identified transcription factors.

The CRISPR/Cas9 knockout strain of the BmKRP of the silkworm is successfully constructed, the influence of a new transcription factor BmKRP on the reproductive process of a female silkworm is preliminarily researched, and the influence is linked with the regulation of BmKr-h1, so that the understanding of BmKr-h1 on the reproductive and egg maturation regulation of the silkworm is enriched, the explanation of a molecular mechanism for regulating the reproductive by silkworm hormone (20E → BmKRP → BmKr-h1) is of great significance, clues and theoretical guidance are provided for the utilization of beneficial insects and new targets and new strategies for pest control, and the application significance is achieved.

While the invention has been disclosed with reference to specific embodiments, it will be apparent that other embodiments and variations of the invention may be devised by those skilled in the art without departing from the true spirit and scope of the invention, and it is intended that the following claims be interpreted to include all such embodiments and equivalent variations. In addition, the contents of all references cited herein are hereby incorporated by reference.

SEQUENCE LISTING

<110> university of south China

Application of <120> KRP gene in pest control

<130> 111

<160> 18

<170> PatentIn version 3.5

<210> 1

<211> 5774

<212> DNA

<213> Bombyx mori

<400> 1

atacaattca ttgcttgtca ttttagccat catttcttta tttggtttaa aattagcaca 60

attacccgct tcatttaaaa tatgttgttt ggccagtaaa gtaccattga tactttcatt 120

ggataaacta tttctaattt tagtctaaat taagtttact tggctaaaaa ttctttcaca 180

atcagctcta gaatgcggta aaattaataa attcaatgaa aattgggata tagacttata 240

cctaatcttc aaaatgtcta ttaacatcgc ctcaaaactt atcagggctt gttgtcaaat 300

taatcttttt atataagaat gagattaatt aaattgttta atacatgaaa ttttaattgc 360

tctattatca tttttcattg acgtgaacaa ataaatataa attaattata ttaaaaacgc 420

atactaaatg aaccagaacc agataattgt tgatttatgt cagcgcgaaa agcattagca 480

gataaggaag gatttctcaa gctcacagtg gaaataagtt cgaaaaatac aaatttaatg 540

cgcaaaactc catctgacga cactaagctt gcctgtgtag gtatttgtct atggtcggcg 600

cagattaagc acaagaaccc aaaaaataca attgtttttt tttgtcaaat gcttgttttg 660

tttgtgaaat acggctacat gttgtcaact ttcggcccgc ttagcaccag ttgggcagtg 720

tactcgttag accatttata gtcctagtga aaaaaaatga tgcatcatca ccatagaatt 780

gatgctgaga ataattaaat tgctcctgta ctgtgtagag tgcatgatct tctatccttg 840

ttcaacttcc taaactgtga ctttgttttc tctttcacct gtccgtaagc gttaacgtca 900

caaatccgtt ccattgttga cctgaatcta tggaacgttt tcaagattgt tcgcgtcgct 960

tgaacagtga aaaaaataca cttttatgac ctaattgaaa cgaatgctga acataaaatg 1020

ggttcaaaga taccaggaaa gttgagaagt caacggttga cgtgtaaatg tgatacttgt 1080

gctctaggat tcaaaaatgt gcaagcgtta cgttcgcacc aagccgttgc ccatccgctg 1140

aagaaaagga ttgcataccg acctaaaacc gaagttatag ccaaaaggaa gcttataccc 1200

cataaaaagg taataaaagt ggcccataaa atgaacaaaa catccgaaaa tgcttcgaca 1260

aaaaaatcac agccacacaa tttattaaga aaatacaaca ctaaaacgcc aatacctagt 1320

gaagacggga aacaatccga atttgagtgc ccggtttgtt ctaaaatatt caaggtttac 1380

tcctgggctt ggaaacacat ccagaaatat cattgcattg acgagaaagg gaaccctgtg 1440

taagtatagt ttcgcgccat aaatttgata caatataaag ttgacgaccc cacgtagttc 1500

tttttaaacc gctgcctgtt atgtagtaat aagtttataa aattaataat ataagcgcgc 1560

ctgcgctttg ctttttgtag taaccagacc aaaatttctc tgaaaaataa gacgattaca 1620

ttgttaatat tacatacagc atttgcgctg tttctgctag caatggacag gatacaatat 1680

aattatggta accagtcaga attgtgttca acagaaaaaa atgtactgat cgaaataact 1740

ctcagttata gcattttttt ttattgtttc tttacaaatg tgtatattaa tttagttcat 1800

tatttgcttt aggacattta tattttattg taataaattc ttaggtgaag taggtctcta 1860

tttaaataga catgaaaagt aagtttactg ccacttggtg acatttggag agcaatttct 1920

cactgcactc atcgtttgct acattatata ggtctgtatt actaaatggc atactatata 1980

agatcaagtg gagacaactt gtgagagccc tatgtaccag cgggataccc taggaccaca 2040

acaacaagaa caagaaagat actacagtgg gagttcaact acacattttt tatacatcat 2100

tagctatgtg taaaaacact ttaattaagt atattgtgtg tgatatcctt cgaaaagaca 2160

ttccctggcc tattaatatt aattctttta gtaactttag tatattctaa acatgtaaat 2220

attctgtgta aaattttatt tggcaatagt atttatcttt aaagtagtta gtccatcata 2280

ttttatttgt ttgtgctatt cttaaaatca aagtttcgcg tatggaattt attcatattt 2340

agtaaatttt ttccctctta ttacctgtca tttttgtgca tactttgata ttggcaaaat 2400

caatgccaaa ggcataggag ccaaattcct atttaaaaaa aagtatattg tgcaaaacca 2460

gcaattttca acagcaaatg tctgaacaaa ttatgattta agttactaca aatattactg 2520

aagtattaga atatttttat aaaacaaatc atgattccca ttagactata gctttaggag 2580

gtaaacaaat ctcacatttt ctatatacca tcctaacaac accatggttt tgcagtttac 2640

caacatcgaa agatataatc aagccagttc gtatagaacg ttgcatcgcc tgtaatgtat 2700

taataaagtc cgatgatcat acatgtggta tatcattctc taacgttctg aagagccagt 2760

actcttgtct cggttgcaat cagcagttca atactttaca tttgtacgaa ctacacatag 2820

caggacttca cagtgaagga gctgagaacc tgttctttcc tgatgaggct ggtgagtttt 2880

attaatgaat aaatttgagt ttagctgtaa tctttcatag gtaacatcat tccttctatt 2940

tcccgggaaa caagaacact tttcagattt atgagtgaca tcagaaaagc ttttgcattg 3000

tgcttttttt ataagcttca gaaccaccca gattgagtaa tagaaagatg aggtatcaaa 3060

ttgatagtgt agcaacctca aagcattaac gttactggtg atgtctggat gtcttgtgag 3120

tccgcacagg tagataccac caccctgcct atttctgacg tgaagcagta atgcgtttcg 3180

tttaaagggt ggcgcagttg ttttaactat acttgagacc ttaaaactta tatctcaagg 3240

tggttggcgt atttacgttg tagatgtata tgggctccag taaccactga acaccaggcg 3300

ggctgtgagc ttgtccaccc aactaagcaa taaaaaatta ctataataaa aatatgaatt 3360

acattgaatg tcaccagcat ataggggatg agtttttaac cacatgctag ttgatttagt 3420

tttagtcatt tttgtttttc tatctaagct gatacctaca cgtaaagggc tcaaacctga 3480

cgacgttgct aacacgatta ctggtgcttg aggtacctca aagcaccgtt agtggatcgg 3540

ggggatccga aatgacgtgt tttgtgcgac gtcgaatgct ttccattcgg tccacagaat 3600

cggaagagta taatagtaca atattattga gggccactaa ttgtatcttt gtaagacatt 3660

tatatcccga cttcatgaat atatggaaac atttctgtaa caatgcaggt aagagagctg 3720

ctagaatgag aaaagcttag tcaaatttat tctgaattaa gaaataaata catatattgt 3780

accaggtatc aatacatata ttgtatcggt aaaatgcaaa taaacacttc aaaatatatt 3840

tttttattga tgtcttatgt tggtgtaaag tttgttatca aaaaaagttt ttaaaactta 3900

tggcgccacg gtaccgaaat gaagtctaca taaaattgct tgttaaaaaa attgtttccc 3960

attacagtca gataaagtat aaattaatta cccacacaaa aatttttaat gataattgtc 4020

gttctgaatt accatattgt aattcatttg aggtttgata gcacacgaac cccattttga 4080

ctggtgctcg taatttcatt acctctttag cacaccgtaa tttttgtgaa aaaaaaagaa 4140

gtgagcgtct cgggaaaccc taccagaagt gataaattaa acaaatcaag ttgaactatt 4200

taatattaaa attatttaac ttgagacaaa gcttaggtta tttcttaaat tttatgtata 4260

ttaatattat aatgtaagga aaactgtaac atacattgaa aaagatacaa aaaatagtta 4320

ggagttacat acgttctcaa tgccaccacc gtgtgcgtga gagaaaggga agccagacag 4380

actggcgccg taacgcgcca cgtaaccaag cgtcttaccc gcccataaga agttattact 4440

tcaacaaaag caagaaaaat attagtacac aagctgtacc cgtccacttc gctgggcatt 4500

taacattaac attattattc ctcaccccca caaagattat catcattgac gccccccaac 4560

tggtgtaggg agtccaacac tcatgtaaat atcagcctat ccattaagta catgtatttt 4620

ctatatggat accaagtttc aagtgaatcg gatgcacggt tcagtagtta taacggaaca 4680

tccgtaaaaa ccactgtagc tttatatatt agtattgatt tatgtattat tgtcgaaatt 4740

taataagcga tttgttgcag cgttctcggc gtggcgacag gacacggaga aacgctgcga 4800

tataaaatac acgaccctaa gcaaaattga taatatgcaa atatatcact gcagtcacat 4860

gaaatgcgac agctctacgg ccagcatgtg cccgtctagc ttcacgaagc aagagttcag 4920

caaaggcata caggtcgtgt actataaagc tcattacggg cacataatcg atgaatacac 4980

cctccccgag gagttcaaga agtactcaat aagctcactg ttgagagata cggattgtta 5040

caccgtgctg agcgtcgact gtgactcgga cgacctcagc gtgcaattca agaggttaat 5100

ggacactatt ttgggtaaat cgttgcatgt taaggagaac gtattgaaat tgttgtacgg 5160

taaagcactg gagatggctt cgctgttgaa cgatgaggct ggctgtaaaa ttgagtgtca 5220

gaatggtaag gagctcgcta aaaagacatc caccgatgac attgtcgagg acgaatctaa 5280

acttgacacg gaaatgaaaa caaaagtgcc cgttaagact tacagcaata cgaggaagaa 5340

cgtgaagttt gaggacaaca aaattgggtc cccgaagttc gggtcgcccg cgtcgctgac 5400

gtcattcaat gattcgtacc ggcacttcgt cgaggacacc ctgggcgcgg tcgacgagaa 5460

aattaaaaag aaaaaaatga tcgttaaaac cagaataggg cagttcaagc ccacctcacc 5520

gacaaacgac agggcgacaa taaaaaaatc gcccaagtct tcaaccaaag ttaaaagtag 5580

cctaagattg aaacgcgact tcgaatacga agtcatagaa agggacaaag aatgcaacat 5640

tatggttata aaaatttaag aaaaaaaaaa aaacaataaa aagcaacttt tcgttgttgt 5700

aaatcaaatt taaattacgc atcgactgaa aagaattaag tttttaaaaa cgtctataaa 5760

ttttgtacaa taaa 5774

<210> 2

<211> 518

<212> PRT

<213> Bombyx mori

<400> 2

Met Gly Ser Lys Ile Pro Gly Lys Leu Arg Ser Gln Arg Leu Thr Cys

1 5 10 15

Lys Cys Asp Thr Cys Ala Leu Gly Phe Lys Asn Val Gln Ala Leu Arg

20 25 30

Ser His Gln Ala Val Ala His Pro Leu Lys Lys Arg Ile Ala Tyr Arg

35 40 45

Pro Lys Thr Glu Val Ile Ala Lys Arg Lys Leu Ile Pro His Lys Lys

50 55 60

Val Ile Lys Val Ala His Lys Met Asn Lys Thr Ser Glu Asn Ala Ser

65 70 75 80

Thr Lys Lys Ser Gln Pro His Asn Leu Leu Arg Lys Tyr Asn Thr Lys

85 90 95

Thr Pro Ile Pro Ser Glu Asp Gly Lys Gln Ser Glu Phe Glu Cys Pro

100 105 110

Val Cys Ser Lys Ile Phe Lys Val Tyr Ser Trp Ala Trp Lys His Ile

115 120 125

Gln Lys Tyr His Cys Ile Asp Glu Lys Gly Asn Pro Val Leu Pro Thr

130 135 140

Ser Lys Asp Ile Ile Lys Pro Val Arg Ile Glu Arg Cys Ile Ala Cys

145 150 155 160

Asn Val Leu Ile Lys Ser Asp Asp His Thr Cys Gly Ile Ser Phe Ser

165 170 175

Asn Val Leu Lys Ser Gln Tyr Ser Cys Leu Gly Cys Asn Gln Gln Phe

180 185 190

Asn Thr Leu His Leu Tyr Glu Leu His Ile Ala Gly Leu His Ser Glu

195 200 205

Gly Ala Glu Asn Leu Phe Phe Pro Asp Glu Ala Ala Phe Ser Ala Trp

210 215 220

Arg Gln Asp Thr Glu Lys Arg Cys Asp Ile Lys Tyr Thr Thr Leu Ser

225 230 235 240

Lys Ile Asp Asn Met Gln Ile Tyr His Cys Ser His Met Lys Cys Asp

245 250 255

Ser Ser Thr Ala Ser Met Cys Pro Ser Ser Phe Thr Lys Gln Glu Phe

260 265 270

Ser Lys Gly Ile Gln Val Val Tyr Tyr Lys Ala His Tyr Gly His Ile

275 280 285

Ile Asp Glu Tyr Thr Leu Pro Glu Glu Phe Lys Lys Tyr Ser Ile Ser

290 295 300

Ser Leu Leu Arg Asp Thr Asp Cys Tyr Thr Val Leu Ser Val Asp Cys

305 310 315 320

Asp Ser Asp Asp Leu Ser Val Gln Phe Lys Arg Leu Met Asp Thr Ile

325 330 335

Leu Gly Lys Ser Leu His Val Lys Glu Asn Val Leu Lys Leu Leu Tyr

340 345 350

Gly Lys Ala Leu Glu Met Ala Ser Leu Leu Asn Asp Glu Ala Gly Cys

355 360 365

Lys Ile Glu Cys Gln Asn Gly Lys Glu Leu Ala Lys Lys Thr Ser Thr

370 375 380

Asp Asp Ile Val Glu Asp Glu Ser Lys Leu Asp Thr Glu Met Lys Thr

385 390 395 400

Lys Val Pro Val Lys Thr Tyr Ser Asn Thr Arg Lys Asn Val Lys Phe

405 410 415

Glu Asp Asn Lys Ile Gly Ser Pro Lys Phe Gly Ser Pro Ala Ser Leu

420 425 430

Thr Ser Phe Asn Asp Ser Tyr Arg His Phe Val Glu Asp Thr Leu Gly

435 440 445

Ala Val Asp Glu Lys Ile Lys Lys Lys Lys Met Ile Val Lys Thr Arg

450 455 460

Ile Gly Gln Phe Lys Pro Thr Ser Pro Thr Asn Asp Arg Ala Thr Ile

465 470 475 480

Lys Lys Ser Pro Lys Ser Ser Thr Lys Val Lys Ser Ser Leu Arg Leu

485 490 495

Lys Arg Asp Phe Glu Tyr Glu Val Ile Glu Arg Asp Lys Glu Cys Asn

500 505 510

Ile Met Val Ile Lys Ile

515

<210> 3

<211> 80

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

taatacgact cactatagga aaatgggttc aaagataccg ttttagagct agaaatagca 60

agttaaaata aggctagtcc 80

<210> 4

<211> 79

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

aaaagcaccg actcggtgcc actttttcaa gttgataacg gactagcctt attttaactt 60

gctatttcta gctctaaaa 79

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

gtactgtgta gagtgcatga 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

aggtattggc gttttagtgt 20

<210> 7

<211> 22

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

cggtgatgac ggtgaaaacc tc 22

<210> 8

<211> 18

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

aagcaccgac tcggtgcc 18

<210> 9

<211> 44

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

taatacgact cactataggg gaagcttata ccccataaaa aggt 44

<210> 10

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 10

tagttcgtac aaatgtaaag tattg 25

<210> 11

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 11

ggaagcttat accccataaa aaggt 25

<210> 12

<211> 44

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 12

taatacgact cactataggt agttcgtaca aatgtaaagt attg 44

<210> 13

<211> 22

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 13

tacggcgtgc agtgcttcag cc 22

<210> 14

<211> 41

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 14

taatacgact cactataggg tgctcaggta gtggttgtcg g 41

<210> 15

<211> 41

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 15

taatacgact cactataggt acggcgtgca gtgcttcagc c 41

<210> 16

<211> 22

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 16

gtgctcaggt agtggttgtc gg 22

<210> 17

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 17

accagaatag ggcagttcaa g 21

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 18

ctttggttga agacttgggc 20

Claims (10)

1. Use of a KRP gene in the preparation of a medicament for regulating the expression of Kr-h1 gene, wherein said KRP gene comprises a nucleotide sequence that specifically binds to ttattata and AATAATCATT in the promoter region of said Kr-h1 gene.
2. 2. The use of claim 1, wherein the KRP gene is BmKRP gene, and the nucleotide sequence of the BmKRP gene is shown in SEQ ID NO: 1 is shown.
3. Use of a KRP gene comprising a nucleotide sequence that specifically binds to ttata and AATAATCATT of the promoter region of the Kr-h1 gene as a suppressor target or silencing target for the preparation of an interfering molecule or a CRISPR/Cas9 system that specifically interferes with the expression of a harmful insect gene or inhibits the growth of a harmful insect.
4. 4. The use of claim 3, wherein the KRP gene is BmKRP gene, and the nucleotide sequence of the BmKRP gene is shown in SEQ ID NO: 1 is shown.
5. 5. The use of claim 3 or 4, wherein the interfering molecule is a dsRNA, an antisense nucleic acid, a small interfering RNA, a microRNA, or a construct capable of expressing or forming said dsRNA, antisense nucleic acid, small interfering RNA, microRNA, wherein the KRP gene or transcript thereof is an inhibitory or silencing target.
6. 6. The use of claim 3 or 4, wherein the CRISPR/Cas9 system comprises Cas9 and a sgRNA that specifically targets the KRP gene; preferably, the nucleotide sequence of sgRNA specifically targeting KRP gene is as set forth in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
7. Use of a KRP gene in the preparation of a formulation for controlling harmful insects, wherein the KRP gene comprises a nucleotide sequence that specifically binds to ttattattaa and AATAATCATT of the promoter region of the Kr-h1 gene; preferably, the KRP gene is BmKRP gene, and the nucleotide sequence of the BmKRP gene is shown in SEQ ID NO: 1 is shown.
8. 8. A method of controlling harmful insects, said method comprising: down-regulating the expression or activity of KRP gene in insect pests; wherein the KRP gene comprises a nucleotide sequence that can specifically bind to TTATTAA and AATAATCATT of the promoter region of the Kr-h1 gene; preferably, the KRP gene is BmKRP gene, and the nucleotide sequence of the BmKRP gene is shown in SEQ ID NO: 1 is shown.
9. 9. The method for controlling harmful insects according to claim 8, wherein the method for down-regulating the expression or activity of KRP gene in harmful insects comprises: knocking out or silencing the KRP gene in the genome of the pest insect; or transferring a down regulator which down-regulates KRP gene transcription, polypeptide expression or polypeptide activity into the body of the insect.
10. 10. A CRISPR/Cas9 system for knocking out a BmKRP gene, comprising Cas9 and sgRNA specifically targeting the BmKRP gene; wherein, the nucleotide sequence of sgRNA of the specific targeting BmKRP gene is shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

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